2.1. Regulation of Eukaryotic DNA Replication
Cell division is a carefully regulated process that involves two events, the duplication of genomic DNA and the physical division of the two daughter cells. Before each new cell division cycle, a decision must be made by the cell of whether to proceed through a new round of DNA replication or withdraw from the cell cycle into a quiescent nonproliferating state. When regulation of this process breaks down the result is uncontrolled cell proliferation which may lead to diseases such as cancer.
Investigators of eukaryotic DNA replication have been faced with the challenge of identifying and characterizing both the specific chromosomal origins of replication and the regulatory proteins that control initiation of replication. Elucidation of the molecular mechanisms regulating DNA replication should lead to the development of therapeutic applications directed at inhibiting DNA replication and cell division.
Studies of DNA replication in mammalian cells has been difficult, due in part to the lack of suitable methods for mapping origins in large and complex genomes. In less complex organisms, such as prokaryotes, yeast and DNA tumor viruses, recent developments have lead to the identification of both cis-acting elements and polypeptides that bind specifically to these elements.
Several proteins from prokaryotes have been characterized which play a role in DNA replication and which bind to single-stranded DNA. The amino acids involved in DNA binding by certain of these proteins have been identified (Gutierrez et al., 1991 J. Biol. Chem. 266, 2104-2111). However, none of these single-stranded DNA binding proteins is known to have any sequence specificity.
In yeast, Saccharomyces cerevisae, cis-acting replication origins, referred to as ARS elements (autonomous replicating sequences), have been identified by their ability to allow extrachromosomal maintenance of plasmids. In general, yeast ARS sequences have a significantly higher A+T content than average chromosomal DNA. Recently a 67 Kd protein, referred to as ACBP (ARS-consensus binding protein) has been purified from yeast cell extracts and shown to bind preferentially to the T-rich single stranded DNA sequences found at the 3' end of the yeast ARS element (Hofmann and Gasser, 1991 Cell 64:951-960).
In a number of DNA tumor viruses, the sequences required for autonomous replication have also been mapped. Perhaps, the best studied of these viruses is Simian Virus 40 (SV40) and a single-stranded DNA-binding protein referred to as RP-Ab is required for replication initiated at the SV40 origin in vitro (Wobbe et al., 1987 Proc. Natl. Acad. Sci. 84:1834-1838; Erdile et al., 1991 J. Biol. Chem. 266:12090-12098). No sequence specificity has been reported for DNA binding by RP-A.
These results raise the possibility that sequence-specific, single-stranded-DNA binding proteins may serve an important function in the initiation of DNA replication. At this time, however, no amino acid sequence has been reported for any mammalian protein known to bind specifically to a single-stranded DNA element.